title: Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.
abstract: Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (Inserm), U964/Centre National deRecherche Scientifique (CNRS), UMR 7104, Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Busso
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
authors.forename: Tatjana
authors.initials: T
authors.lastname: Heidebrecht
authors.forename: Christophe
authors.initials: C
authors.lastname: Romier
authors.forename: Yossi
authors.initials: Y
authors.lastname: Jacobovitch
authors.forename: Ada
authors.initials: A
authors.lastname: Dantes
authors.forename: Loubna
authors.initials: L
authors.lastname: Salim
authors.forename: Edouard
authors.initials: E
authors.lastname: Troesch
authors.forename: Anja
authors.initials: A
authors.lastname: Schuetz
authors.forename: Udo
authors.initials: U
authors.lastname: Heinemann
authors.forename: Gert E
authors.initials: GE
authors.lastname: Folkers
authors.forename: Arie
authors.initials: A
authors.lastname: Geerlof
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.forename: Andrea
authors.initials: A
authors.lastname: Polewacz
authors.forename: Claudia
authors.initials: C
authors.lastname: Quedenau
authors.forename: Konrad
authors.initials: K
authors.lastname: Büssow
authors.forename: Rachel
authors.initials: R
authors.lastname: Adamson
authors.forename: Elena
authors.initials: E
authors.lastname: Blagova
authors.forename: Julia
authors.initials: J
authors.lastname: Walton
authors.forename: Jared L
authors.initials: JL
authors.lastname: Cartwright
authors.forename: Louise E
authors.initials: LE
authors.lastname: Bird
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
authors.forename: Nick S
authors.initials: NS
authors.lastname: Berrow
authors.forename: Keith S
authors.initials: KS
authors.lastname: Wilson
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.forename: Anastassis
authors.initials: A
authors.lastname: Perrakis
authors.forename: Patrick H N
authors.initials: PH
authors.lastname: Celie
mesh_terms: D000047:Academies and Institutes; D023081:CCAAT-Binding Factor / Q000096:biosynthesis / Q000235:genetics; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005060:Europe; D064248:Geminin; D005822:Genetic Vectors / Q000592:standards*; D007391:International Cooperation; D007557:Israel; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D019473:Transcription Factors, TFII / Q000096:biosynthesis / Q000235:genetics
chemical_list: D023081:CCAAT-Binding Factor; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D046912:Multiprotein Complexes; C492061:NFYA protein, human; C492063:NFYB protein, human; C469358:NFYC protein, human; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE